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1.
Torres R Ramón F de la Mata I Acebal C Castillón MP 《Applied microbiology and biotechnology》1999,53(1):81-84
At 28 °C, Streptomyces lavendulae produced high levels of penicillin V acylase (178 IU/l of culture) when grown on skim milk as the sole nutrient source for
275 h. The enzyme showed catabolite repression by glucose and was produced in the stationary phase of growth. Penicillin V
was a good inducer of penicillin V acylase formation, while phenoxyacetic acid, the side-chain moiety of penicillin V, did
not alter enzyme production significantly. The enzyme was stable between pH 6 and 11 and at temperatures from 20 °C to 55 °C.
This extracellular enzyme was able to hydrolyse natural penicillins and unable to hydrolyse penicillin G.
Received: 22 March 1999 / Received revision: 16 June 1999 / Accepted: 20 June 1999 相似文献
2.
Purification and properties of a novel raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus firmus 总被引:3,自引:0,他引:3
B. N. Gawande A. Goel A. Y. Patkar S. N. Nene 《Applied microbiology and biotechnology》1999,51(4):504-509
A novel raw starch degrading cyclomaltodextrin glucanotransferase (CGTase; E.C. 2.4.1.19), produced by Bacillus firmus, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The molecular weight of the
pure protein was estimated to be 78 000 and 82 000 Da, by SDS-PAGE and gel filtration, respectively. The pure enzyme had a
pH optimum in the range 5.5–8.5. It was stable over the pH range 7–11 at 10 °C, and at pH 7.0 at 60 °C. The optimum temperature
for enzyme activity was 65 °C. In the absence of substrate, the enzyme rapidly lost its activity above 30 °C. K
m and k
cat for the pure enzyme were 1.21 mg/ml and 145.17 μM/mg per minute respectively, with soluble starch as the substrate. For cyclodextrin
production, tapioca starch was the best substrate used when gelatinized, while wheat starch was the best substrate used when
raw. This CGTase could degrade raw wheat starch very efficiently; up to 50% conversion to cyclodextrins was obtained from
150 g/l starch without using any additives. The enzyme produced α-, β- and γ-cyclodextrins in the ratio of 0.2:9.2:0.6 and
0.2:8.6:1.2 from gelatinized tapioca starch and raw wheat starch with 150 g/l concentration respectively, after 18 h incubation.
Received: 25 September 1998 / Received revision: 15 December 1998 / Accepted: 21 December 1998 相似文献
3.
Pi Nyvall Jerome Pelloux Howard V. Davies Marianne Pedersén Roberto Viola 《Planta》1999,209(1):143-152
Red algae (Rhodophyceae) are photosynthetic eukaryotes that accumulate starch granules in the cytosol. Starch synthase activity
in crude extracts of Gracilaria tenuistipitata Chang et Xia was almost 9-fold higher with UDP[U-14C]glucose than with ADP[U-14C]glucose. The activity with UDP[U-14C]glucose was sensitive to proteolytic and oxidative inhibition during extraction whilst the activity with ADP[U-14C]glucose appeared unaffected. This indicates the presence of separate starch synthases with different substrate specificities
in G. tenuistipitata. The UDPglucose: starch synthase was purified and characterised. The enzyme appears to be a homotetramer with a native Mr of 580 kDa and displays kinetic properties similar to other α-glucan synthases such as stimulation by citrate, product (UDP)
inhibition and broad primer specificity. We propose that this enzyme is involved in cytosolic starch synthesis in red algae
and thus is the first starch synthase described that utilises UDPglucose in vivo. The biochemical implications of the different
compartmentalisation of starch synthesis in red algae and green algae/plants are also discussed.
Received: 29 January 1999 / Accepted: 11 March 1999 相似文献
4.
A highly thermostable endo-(1,4)-β-mannanase from the marine bacterium Rhodothermus marinus 总被引:2,自引:0,他引:2
Rhodothermus marinus ATCC 43812, a thermophilic bacterium isolated from marine hot springs, possesses hydrolytic activities for depolymerising
substrates such as carob-galactomannan. Screening of expression libraries identified mannanase-positive clones. Subsequently,
the corresponding DNA sequences were determined, eventually identifying a coding sequence specifying a 997 amino acid residue
protein of 113 kDa. Analyses revealed an N-terminal domain of unknown function and a C-terminal mannanase domain of 550 amino
acid residues with homology to known mannanases of glycosidase family 26. Action pattern analysis categorised the R. marinus mannanase as an endo-acting enzyme with a requirement for at least five sugar moieties for effective catalytic activity.
When expressed in Escherichia coli, purified gene product with catalytic activity was mainly found as two protein fragments of 45 kDa and 50 kDa. The full-length
protein of 113 kDa was only detected in crude extracts of R. marinus, while truncated protein-containing fractions of the original source resulted in a major active protein of 60 kDa. Biochemical
analysis of the mannanase revealed a temperature and pH optimum of 85 °C and pH 5.4, respectively. Purified, E. coli-produced protein fragments showed high heat stability, retaining more than 70% and 25% of the initial activity after 1 h
incubation at 70 °C and 90 °C, respectively. In contrast, R. marinus-derived protein retained 87% activity after 1 h at 90 °C. The enzyme hydrolysed carob-galactomannan (locust bean gum) effectively
and to a smaller extent guar gum, but not yeast mannan.
Received: 5 November 1999 / Received revision: 19 January 2000 / Accepted: 23 January 2000 相似文献
5.
A number of nutritional factors influencing growth and glucose oxidase (EC 1.1.3.4) production by a newly isolated strain
of Penicillium pinophilum were investigated. The most important factors for glucose oxidase production were the use of sucrose as the carbon source,
and growth of the fungus at non-optimal pH 6.5. The enzyme was purified to apparent homogeneity with a yield of 74%, including
an efficient extraction step of the mycelium mass at pH 3.0, cation-exchange chromatography and gel filtration. The relative
molecular mass (M
r) of native glucose oxidase was determined to be 154 700 ± 4970, and 77 700 for the denatured subunit. Electron-microscopic
examinations revealed a sandwich-shaped dimeric molecule with subunit dimensions of 5.0 × 8.0 nm. Glucose oxidase is a glycoprotein
that contains tightly bound FAD with an estimated stoichiometry of 1.76 mol/mol enzyme. The enzyme is specific for d-glucose, for which a K
m value of 6.2 mM was determined. The pH optimum was determined in the range pH 4.0–6.0. Glucose oxidase showed high stability
on storage in sodium citrate (pH 5.0) and in potassium phosphate (pH 6.0), each 100 mM. The half-life of the activity was
considerably more than 305 days at 4 °C and 30 °C, and 213 days at 40 °C. The enzyme was unstable at temperatures above 40 °C
in the range pH 2.0–4.0 and at a pH above 7.0.
Received: 18 November 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997 相似文献
6.
K. Hofvendahl E. W. J. van Niel B. Hahn-Hägerdal 《Applied microbiology and biotechnology》1999,51(5):669-672
Lactococcus lactis ssp. lactis ATCC 19435 is known to produce mixed acids when grown on maltose. A change in fermentation conditions only, elevated temperatures
(up to 37 °C) and reduced pH values (down to 5.0) resulted in a shift towards homolactic product formation. This was accompanied
by decreased growth rate and cell yield. The results are discussed in terms of redox balance and maintenance, and the regulation
of lactate dehydrogenase and pyruvate formate-lyase.
Received: 14 December 1998 / Received revision: 12 January 1999 / Accepted: 22 January 1999 相似文献
7.
A Gram-negative bacterium producing a heat-stable nitrilase highly active on aliphatic dinitriles 总被引:8,自引:0,他引:8
J. E. Gavagan R. DiCosimo A. Eisenberg S. K. Fager P. W. Folsom E. C. Hann K. J. Schneider R. D. Fallon 《Applied microbiology and biotechnology》1999,52(5):654-659
A Gram-negative bacterial strain, identified as Acidovorax facilis strain 72W, has been isolated from soil by enrichment using 2-ethylsuccinonitrile as the sole nitrogen source. This strain
grows on a variety of aliphatic mono- and dinitriles. Experiments using various heating regimes indicate that nitrile hydratase,
amidase and nitrilase activities are present. The nitrilase is efficient at hydrolyzing aliphatic dinitriles to cyanoacid
intermediates. It has a strong bias for C3–C6 dinitriles over mononitriles of the same chain length. Whole, resting cell hydrolysis of 2-methylglutaronitrile results in
4-cyanopentanoic acid and 2-methylglutaric acid as the major products. Heating, at least 20 min at 50 °C, eliminates nitrile
hydratase and amidase activities, resulting in greater than 97% selectivity to 4-cyanopentanoic acid. The nitrilase activity
has good heat stability, showing a half-life of 22.7 h at 50 °C and a temperature optimum of at least 65 °C for activity.
The strain has been deposited as ATCC 55746.
Received: 26 January 1999 / Received revision: 10 June 1999 / Accepted: 27 June 1999 相似文献
8.
A. E. Cazemier J. C. Verdoes H. J. M. Op den Camp J. H. P. Hackstein A. J. J. van Ooyen 《Applied microbiology and biotechnology》1999,52(2):232-239
A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA
fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A
had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50–55 °C. The recombinant endoglucanase
Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding
domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262–319 and 448–473,
which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding.
Received: 14 January 1999 / Received revision: 29 March 1999 / Accepted: 6 April 1999 相似文献
9.
P. Wahl P. Walser-Volken K. Laumen M. Kittelmann O. Ghisalba 《Applied microbiology and biotechnology》1999,53(1):12-18
Rhodococcus globerulus K1/1 was found to express an inducible (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed
at about pH 6.5. Purification of the enzyme to a single band in a Coomassie blue-stained SDS-PAGE gel was achieved by nucleic
acid and ammonium sulfate precipitation of Rhodococcus globerulus K1/1 crude extract and column chromatography on TSK Butyl-650(S) Fractogel and Superose 12HR. The amidohydrolase was purified to a homogeneity leading to a tenfold increase of the specific
activity with a recovery rate of 65%. At pH 7.0 and 23 °C the enzyme showed no loss of activity after 30 days incubation.
The amidohydrolase was stable up to 55 °C. The enzyme was inhibited strongly only by 10 mM Zn2+ among the tested metal cations and was inhibited 100% by 0.01 mM phenylmethanesulfonyl fluoride. The molecular weight of
the native enzyme was estimated to be 92 kDa by gel filtration and 55 kDa by SDS-PAGE, suggesting a homodimeric structure.
Received: 8 February 1999 / Received revision: 3 May 1999 / Accepted: 7 May 1999 相似文献
10.
The extremely thermophilic archaeon Thermococcus hydrothermalis, isolated from a deep-sea hydrothermal vent in the East Pacific Rise at 21°N, produced an extracellular pullulanase. This
enzyme was purified 97-fold to homogeneity from cell-free culture supernatant. The purified pullulanase was composed of a
single polypeptide chain having an estimated molecular mass of 110 kDa (gel filtration) or 128 kDa (sodium dodecyl sulfate/polyacryl
amide gel electrophoresis). The enzyme showed optimum activity at pH 5.5 and 95 °C. The thermostability and the thermoactivity
were considerably increased in the presence of Ca2+. The enzyme was activated by 2-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and α-cyclodextrin were inhibitors. This enzyme was able to hydrolyze, in addition to the α-1,6-glucosidic
linkages in pullulan, α-1,4-glucosidic linkages in amylose and soluble starch, and can therefore be classified as a type II
pullulanase or an amylopullulanase. The purified enzyme displayed Michaelis constant (K
m) values of 0.95 mg/ml for pullulan and 3.55 mg/ml for soluble starch without calcium and, in the presence of Ca2+, 0.25 mg/ml for pullulan and 1.45 mg/ml for soluble starch.
Received: 19 November 1997 / Received revision: 9 March 1998 / Accepted: 14 March 1998 相似文献
11.
Glucose oxidase from Penicillium amagasakiense was purified to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas
chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type
carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety.
A molecular mass of 130 kDa (gel filtration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE)
for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not been published for glucose oxidase from
P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not affect the kinetics
of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic
degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of
both enzymes was shown to be dependent on the buffer concentration and was enhanced by certain additives, particularly 1 M
(NH4)2SO4, which stabilised glucose oxidase 100- to 300-fold at 50 °C and pH 7–8, and 2 M KF, which stabilised the enzyme up to 36-fold
at 60 °C and pH 6. In sodium acetate buffer, changes in pH (4–6) affected the affinity for glucose but had no effect on the
V
max of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in V
max and a 2-fold decrease in K
m were observed.
Received: 8 October 1996 / Received revision: 14 January 1997 / Accepted: 17 January 1997 相似文献
12.
Influence of complex nutrients, temperature and pH on bacteriocin production by Lactobacillus sakei CCUG 42687 总被引:1,自引:0,他引:1
Aasen IM Møretrø T Katla T Axelsson L Storrø I 《Applied microbiology and biotechnology》2000,53(2):159-166
The effects of process conditions and growth kinetics on the production of the bacteriocin sakacin P by Lactobacillus sakei CCUG 42687 have been studied in pH-controlled fermentations. The fermentations could be divided into phases based on the
growth kinetics, phase one being a short period of exponential growth, and three subsequent ones being phases of with decreasing
specific growth rate. Sakacin P production was maximal at 20 °C. At higher temperatures (25–30 °C) the production ceased at
lower cell masses, when less glucose was consumed, resulting in much lower sakacin P concentrations. With similar media and
pH, the maximum sakacin P concentration at 20 °C was seven times higher than that at 30 °C. The growth rate increased with
increasing concentrations of yeast extract, and the maximum concentration and specific production rate of sakacin P increased
concomitantly. Increasing tryptone concentrations also had a positive influence upon sakacin P production, though the effect
was significantly lower than that of yeast extract. The maximum sakacin P concentration obtained in this study was 20.5 mg
l−1. On the basis of the growth and production kinetics, possible metabolic regulation of bacteriocin synthesis is discussed,
e.g. the effects of availability of essential amino acids, other nutrients, and energy.
Received: 7 June 1999 / Received revision: 15 September 1999 / Accepted: 17 September 1999 相似文献
13.
The amylopullulanse produced by Bacillus sp. DSM 405 was purified to homogeneity. It exhibited dual activity, cleaving the α1-4 bonds in starch, releasing a range
of malto-oligosaccharides, and also cleaving the α1-6 bonds in pullulan, releasing maltotriose as the sole end-product. The
enzyme was a glycoprotein and had a relative molecular mass of 126 000 and an isoelectric point of 4.3. While the enzyme was
optimally active on starch at pH 6.5 and at pH 6.0 on pullulan, activity on both substrates was maximal at 70 °C. Kinetic
analyses of the enzyme in a system that contained both starch and pullulan as two competing substrates demonstrated the dual
specificity of the enzyme. Chemical modification of the carboxyl groups within the active centre of the protein showed that
one active site was responsible for hydrolysis of the α1-4 and α1-6 bonds in starch and pullulan respectively. This is the
first comprehensive investigation of an amylopullulanse produced by an aerobic bacterium, showing a single active site responsible
for both activities.
Received: 3 August 1998 / Received revision: 13 October 1998 / Accepted: 16 October 1998 相似文献
14.
Bacillus subtilis CBTK 106, isolated from banana wastes, produced high titres of α-amylase when banana fruit stalk was used as substrate in
a solid-state fermentation system. The effects of initial moisture content, particle size, cooking time and temperature, pH,
incubation temperature, additional nutrients, inoculum size and incubation period on the production of α-amylase were characterised.
A maximum yield of 5 345 000 U mg-1 min-1 was recorded when pretreated banana fruit stalk (autoclaved at 121 °C for 60 min) was used as substrate with 70% initial
moisture content, 400 μm particle size, an initial pH of 7.0, a temperature of 35 °C, and additional nutrients (ammonium sulphate/sodium
nitrate at 1.0%, beef extract/peptone at 0.5%, glucose/sucrose/starch/maltose at 0.1% and potassium chloride/sodium chloride
at 1.0%) in the medium, with an inoculum-to-substrate ratio of 10% (v/w) for 24 h. The enzyme yield was 2.65-fold higher with
banana fruit stalk medium compared to wheat bran.
Received: 18 April 1995/Received last revison: 6 May 1996/Accepted: 9 May 1996 相似文献
15.
A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA 总被引:10,自引:0,他引:10
An improved method for the electrotransformation of wild-type Corynebacterium glutamicum (ATCC 13032) is described. The two crucial alterations to previously developed methods are: cultivation of cells used for
electrotransformation at 18 °C instead of 30 °C, and application of a heat shock immediately following electrotransformation.
Cells cultivated at sub optimal temperature have a 100-fold improved transformation efficiency (108 cfu μg−1) for syngeneic DNA (DNA isolated from the same species). A heat shock applied to these cells following electroporation improved
the transformation efficiency for xenogeneic DNA (DNA isolated from a different species). In combination, low cultivation
temperature and heat shock act synergistically and increased the transformation efficiency by four orders of magnitude to
2.5 × 106 cfu μg−1 xenogeneic DNA. The method was used to generate gene disruptions in C. glutamicum.
Received: 26 March 1999 / Received revision: 9 June 1999 / Accepted: 11 June 1999 相似文献
16.
K. Hofvendahl C. Åkerberg G. Zacchi B. Hahn-Hägerdal 《Applied microbiology and biotechnology》1999,52(2):163-169
Simultaneous saccharification of starch from whole-wheat flour and fermentation to lactic acid (SSF) was investigated. For
saccharification the commercial enzyme mixture SAN Super 240 L, having α-amylase, amyloglucosidase and protease activity,
was used, and Lactococcus lactis ssp. lactis ATCC 19435 was used for the fermentation. SSF was studied at flour concentrations corresponding to starch concentrations
of 90 g/l and 180 g/l and SAN Super concentrations between 3 μl/g and 8 μl/g starch. Kinetic models, developed for the saccharification
and fermentation, respectively, were used for simulation and data from SSF experiments were used for model verification. The
model simulated SSF when sufficient amounts of nutrients were available during fermentation. This was achieved with high wheat
flour concentrations or with addition of yeast extract or amino acids. Nutrient release was dependent on the level of enzyme
activity.
Received: 26 January 1999 / Accepted: 20 February 1999 相似文献
17.
H. E. M. Mc Mahon C. T. Kelly W. M. Fogarty 《Applied microbiology and biotechnology》1997,48(4):504-509
The α-amylase of Streptomyces sp. IMD 2679 was subject to catabolite repression. Four different growth rates were achieved when the organism was grown
at 40 °C and 55 °C in the presence and absence of cobalt, with an inverse relationship between α-amylase production and growth
rate. Highest α-amylase yields (520 units/ml) were obtained at the lowest growth rate (0.062 h−1), at 40 °C in the absence of cobalt, while at the highest growth rate (0.35 h−1), at 55 °C in the presence of cobalt, α-amylase production was decreased to 150 units/ml. As growth rate increased, the rate
of specific utilisation of the carbon source maltose also increased, from 46 to 123 μg maltose (mg biomass)−1 h−1. The pattern and levels of α-glucosidase (the enzyme degrading maltose) detected intracellularly in each case, indicate that
growth rate effectively controls the rate of feeding of glucose to the cell, and thus catabolite repression.
Received: 17 February 1997 / Received revision: 29 April 1997 / Accepted: 11 May 1997 相似文献
18.
R Rech C F Cassini A Secchi M A Z Ayub 《Journal of industrial microbiology & biotechnology》1999,23(2):91-96
We studied the utilization of protein-hydrolyzed sweet cheese whey as a medium for the production of β-galactosidase by the
yeasts Kluyveromyces marxianus CBS 712 and CBS 6556. The conditions for growth were determined in shake cultures. The best growth occurred at pH 5.5 and
37°C. Strain CBS 6556 grew in cheese whey in natura, while strain CBS 712 needed cheese whey supplemented with yeast extract. Each yeast was grown in a bioreactor under these
conditions. The strains produced equivalent amounts of β-galactosidase. To optimize the process, strain CBS 6556 was grown
in concentrated cheese whey, resulting in a higher β-galactosidase production. The β-galactosidase produced by strain CBS
6556 produced maximum activity at 37°C, and had low stability at room temperature (30°C) as well as at a storage temperature
of 4°C. At −4°C and −18°C, the enzyme maintained its activity for over 9 weeks.
Received 20 January 1999/ Accepted in revised form 30 April 1999 相似文献
19.
S Hayashi S Sako H Yokoi Y Takasaki K Imada 《Journal of industrial microbiology & biotechnology》1999,22(3):160-163
β-Glucosidase hydrolyzing cellobiose was extracted from Aureobasidium sp ATCC 20524 and purified to homogeneity. The molecular mass was estimated to be about 331 kDa. The enzyme contained 26.5%
(w/w) carbohydrate. The optimum pH and temperature for the enzyme reaction were pH 4 and 80°C, respectively. The enzyme was
stable at a wide range of pH, 2.2–9.8, after 3 h and at 75°C for 15 min. The kinetic parameters were determined. The enzyme
was relatively stable against typical organic enzyme inhibitors. The enzyme also hydrolyzed gentiobiose, p-nitrophenyl-β-glucoside and salicin.
Received 05 November 1998/ Accepted in revised form 14 February 1999 相似文献
20.
B. L. García A. S. Ball J. Rodríguez M. I. Pérez-Leblic M. E. Arias J. L. Copa-Patiño 《Applied microbiology and biotechnology》1998,50(2):213-218
Streptomyces avermitilis CECT 3339 produces extracellular ferulic acid esterase (FAE) activity during growth on a range of lignocellulose substrates.
Maximal levels of FAE activity were detected in culture filtrates from S. avermitilis CECT 3339 grown in media containing wheat bran and yeast extract as carbon and nitrogen sources respectively. Biochemical
characterization of this enzyme activity revealed that it was 100-fold higher when wheat bran was pretreated with Celluclast
(a mix of hydrolytic enzymes). FAE was found to be end-product-inhibited. Characterization of the properties of the enzyme
showed that FAE exhibited an activity optimum pH at 6 with pH stability between pH 6 and 8. The optimum temperature was 50 °C
while the temperature stability was between 30 °C and 40 °C, with rapid inactivation at 60 °C and above. The characteristics
and stability of FAE from S. avermitilis CECT 3339 suggest a potential role for this enzyme in combination with endoxylanases for the upgrading of plant-residue silage
and for biopulping.
Received: 17 November 1997 / Received revision: 13 March 1998 / Accepted: 13 April 1998 相似文献